Cells undergoing programmed cell-death (known as apoptosis) are rapidly removed by monocyte-derived macrophages. This phenomenon infers that apoptotic cells might secrete specific factors and these factors cause chmotaxis to leucocyte and monocyte. The endothelial monocyte-activating polypeptide II (hereinafter, referred to as “EMAP II”) is known as one of the specific factors because it is released from the apoptotic cells and causes chemotaxis.
The EMAP II is the C-terminal domain of the p43 protein—the precursor of the EMAP II, consisting of 312 amino acids—and it is cleaved to form by caspase-7, in which aspartic acid—the 146th amino acid of p43 protein—is activated in apoptotic cells (Quevillion, S. et al., J. Biol. Chem., 272:32573–32579, 1997; Behrensdorf, M. A. et al., FEBS Lett., 466:143–147, 2000). The structure and maturation of the EMAP II is similar to that of an IL-1β, a cytokine involved in a proinflammatory response, and 14.5 kDa of the IL-1β is cleaved to form from 33 kDa of inactive pre-IL-1β by ICE (caspase-1). An EMAP II is the mediator of proinflammatory response that induces the expression of tissue factor, tumor necrosis factor (hereinafter, referred to as “TNF”) and interleukin-8 (hereinafter, referred to as “IL-8”) in mononuclear phagocyte and polymorphonuclear leucocytes. Also, in a tissue expressing a high level of EMAPII mRNA, macrophages are accumulated. This means that an EMAP II is a chemotaxis material leading macrophage to dead cells. It is known that the EMAP II acts as a cytokine, and the 15 amino acids of the N-terminal domain of an EMAP II play a vital role in the reaction (Quevillon, S. et al., J. Biol. Chem., 272:32573–32579, 1997; Kao, J. et al., J. Biol. Chem., 269:9774–7982, 1994; Kao, J. et al., J. Biol. Chem., 267:20239–20247, 1992; Kao, J. et al., J. Biol. Chem., 269:25106–25119, 1994; Knies, U. E. et al., PNAS USA, 95:12322–12327, 1998). In the U.S. Pat. No. 5,641,867, it is described that the N-terminal domain of an EMAP II that comprises arginine-isoleucine-glycine-arginine-isoleucine-threonine is an important residue in cytokine function of the EMAP II. Recently, it was reported that the EMAP II repressed the growth of primary and metastatic tumor in proliferating endothelial cells that are not causing particular side-effect in normal cell (Schwarz, M. A. et al., J. Exp. Med., 190:341–353, 1999).
On the other hand, the p43 protein is expressed extensively. The expression level of p43 protein varies temporally and spatially, especially in a developing mouse. For example, it was shown that the expression of a p43 in the lung of mouse from 8 days to 16 days after its birth was increased dramatically. In addition, the p43 is highly expressed in the microglial cells in the lesions of autoimmune disease such as encephalomyelitis, neuritis and uveitis. The high expression level of the p43 in specific developmental stage and tissues suggests that the p43 could have unexpected functions in angiogenesis, inflammation, and apoptosis (Tas, M. P. R., and Marray, J. C., Int. J. Biochem. Cell. Biol., 28:837–841, 1996; Schwarz, M. J. et al., Glia, 20:365–372, 1997; Schuesner, H. J. et al., Glia, 20:365–372, 1997; Berger, A. C. et al., J. Immunother., 23:519–527, 2000).
As mentioned previously, the EMAP II—C-terminal domain of p43—has been studied extensively for its cytokine activities; however, p43 (i.e. pro-EMAP II) and its N-terminal domain have not been understood. Therefore, the present inventors have studied to disclose that a p43 can act as a more effective cytokine and as an immunological enhancement agent than the EMAP II. We did this by comparing cytokine activity of a p43 with that of an EMAP II, C-terminal domain of p43 in the PCT application No. PCT/KR00/00630.
The present inventors compared the secretion pattern of a p43 with that of an EMAP II in normal cells and apoptotic cells, and reported that the cytokine acting in physiological condition was the p43 and not the EMAP II. Since the EMAP II is secreted during the late stage of apoptosis in which cells are completely destroyed in normal cells, it is not active in the early stage of apoptosis; whereas, the p43 is constitutively secreted from various cells irrespectively of apoptosis (Ko Y G et al., A cofactor of tRNA synthetase, p43, is secreted to up-regulate proinflammatory genes, J. Biol. Chem. 2001, Apl 5, 276).
While studying the p43, the present inventors achieved the present invention by discovering that the peptides, including N-terminal domain of p43, showed an excellent cytokine activity with the result of estimating cytokine activities of deletion-mutants of the p43 protein.